Let's first turn on the cache for increased performance and improved styling

# Set some global knitr options
library("knitr")
opts_chunk$set(tidy=TRUE, tidy.opts=list(blank=FALSE, width.cutoff=60), cache=TRUE, messages=FALSE)

• Remember that Affymetrix and Illumina use multiple probes for a given gene
• So it is important to summarize these "replicated" measurements before any down stream analysis
• There exist many different ways to do this for both Affymetrix and Illumina

• ~20 probes that “perfectly” represent the gene (Perfect Match)

• ~20 probes that do not match the gene sequence (Mismatch)

• Probeset

For a valid gene expression measurement the Perfect Match sticks and the Mistach does not!

Probe summary - MAS 4.0

Is this an appropriate model?

Li, C., & Wong, W. H. (2001). Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biology.

The same can be done with the probes by looking at the variance of \(\phi\)

Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B., & Speed, T. P. (2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Research, 31(4), e15.

Irizarry et al. (2003) argued that MM is a poor measure of non specific hybridization

• Modeling \(PM-MM\) results in large variance!
• Model only the PM intensities
• Before do a "gentle" background correction

\[\log_2(PM^*) =\theta_i+\phi_j+\epsilon_{ij}\]

Where \(PM^*\) is a normalized background corrected intensity

This the basic model implemeted in the robust multiarray average RMA

This can also be used in new Affymetrix arrays that have no MM probes

• Does the sequence composition have an effect?
  • Yes
• Should we account for it?
  • How?
• The physical system producing probe intensity is a complicated one!
• Non specific background hybridization is related to the base compositions.
• Many factors (nucleotide composition, base position, neighbors?, etc)
  • G/C content

Naef, F., & Magnasco, M. O. (2003). Solving the riddle of the bright mismatches: labeling and effective binding in oligonucleotide arrays. Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics, 68(1 Pt 1), 011906.

Wu, Z., & Irizarry, R. A. (2004). Stochastic models inspired by hybridization theory for short oligonucleotide arrays. the eighth annual international conference (pp. 98–106). New York, New York, USA: ACM. doi:10.1145/974614.974628

• There exist many different algorithms for computing probe summaries from Affymetrix arrays
• RMA is a popular package for probe summary
• Other good packages available in Bioconductor including gcRMA, PUMA, BGX, PLIER, XPS, etc
• Some of the packages are optimized for large datasets

Bead level data from Illumina BeadArrays involve an analysis similar to Affymetrix arrays to obtain probe summary data.

Ritchie, M. E., Dunning, M. J., Smith, M. L., Shi, W., & Lynch, A. G. (2011). BeadArray expression analysis using bioconductor. PLoS Computational Biology, 7(12), e1002276. doi:10.1371/journal.pcbi.1002276

Dunning, M. J., Barbosa-Morais, N. L., Lynch, A. G., Tavaré, S., & Ritchie, M. E. (2008). Statistical issues in the analysis of Illumina data. BMC Bioinformatics, 9(1), 85. doi:10.1186/1471-2105-9-85

Conclusions from the paper:

• Access to bead level data promotes more detailed quality assessment and more flexible analyses. Bead level data can be summarised on a relevant scale. We were able to use the means and variances of the log2 data in the DE analysis to improve our ability to detect known changes in the spikes.

• The background correction and summarisation methods used in BeadStudio reduce bias and produce robust gene expression measurements. However, we find that back- ground normalisation introduces a significant number of negative values and much increased variability.

• Base composition of probes has an effect on intensity and further investigation is required to remove this effect.